Gene therapy broadly refers to the transfer of genetic material into cells and the expression of that material in those cells for a therapeutic purpose. The goal is to produce the desired protein in the appropriate quantity and the proper location. Although a variety of methods have been developed to deliver therapeutic nucleic acids to cells, many of these methods are limited by relatively inefficient transfer of the therapeutic nucleic acid to the target cells. Because viruses are highly efficient at infecting susceptible cells, viruses are now recognised as being useful vehicles for the transfer of therapeutic nucleic acids into cells for the purpose of gene therapy.
Viruses fall broadly into two distinct groups: those that integrate into the genome of transduced cells and those that do not. An integrating virus inserts its viral genome into host DNA to facilitate long-term gene expression. For a non-integrating virus, however, the viral genome exists extra-chromosomally as an episome in the nucleus of transduced cells. Depending on the ability of the virus to replicate, the viral genome is either passed on faithfully to every daughter cell or is eventually lost during cell division.
Retroviruses and adeno-associated viruses (AAVs) may integrate into the host DNA to provide a steady level of expression following transduction and incorporation into the host genome. As the target DNA is replicated, so too is the inserted therapeutic gene embedded in the transferred chromosomal DNA. Thus, transduction via these vectors can produce durable gene expression. This can be advantageous in tumour vaccine strategies in which a steady level of gene expression may enhance efficacy.
In contrast, adenovirus and vaccinia virus vectors do not integrate into the host DNA but exist as episomes. Thus, a transferred gene is expressed without actual integration of the gene into the target cell genome. Generally, non-integrating viruses are used when transient gene expression is desired.
Examples of viruses that may be used to deliver nucleic acids to cells for gene therapy purposes include adenovirus, adeno-associated virus (AAV), retrovirus, herpes simplex virus, vaccinia virus, poliovirus, sindbis virus, HIV-1, avian leukosis virus, sarcoma virus, Epstein-Barr virus, papillomavirus, foamy virus, influenza virus, Newcastle disease virus, sendai virus, lymphocytic choriomeningitis virus, polyoma virus, reticuloendotheliosis virus, Theiler's virus, and other types of RNA and DNA viruses.
The use of attenuated and killed viruses for purposes of vaccination is also well known. In addition, viruses are also becoming increasingly important as tools for research and diagnostics. The increasing importance of viruses as tools for gene therapy, vaccination, and research and diagnosis has led to a need to develop viral compositions that may be manufactured, stored and used without compromising viral efficacy. For example, viral compositions for vaccination must be able to maintain the immunogenicity of a virus, or the immunogenicity of a component of the virus. In the case of compositions of viruses to be used for gene therapy, it is critical that the efficacy of the live viral formulations carrying therapeutic transgenes be maintained.
Because viruses are biological entities consisting of a nucleic acid encapsulated by a protein coat, they are susceptible to the same chemical and physical processes that may degrade or inactivate proteins and nucleic acids. In particular, live viruses may often be very susceptible to damage, as any change in the conformation or integrity of one or more components of the virus coat or the encapsulated nucleic acid may lead to a loss of infectivity. As such, biopharmaceutical products containing compositions of viruses for vaccination or gene therapy usually require stringent conditions to avoid physicochemical degradation and to maintain biological activity. Degradation of viruses in such compositions may occur during isolation, production, purification, formulation, storage, shipping or delivery of the virus. Accordingly, biopharmaceutical compositions of viruses must be formulated to provide protection of the virus against factors such as temperature, pH, pressure, oxidising agents, ionic content, light, radiation, ultrasound, and changes in phase (for example as occurs during freezing and thawing (“freeze-thawing”).
In addition to the factors already discussed, other factors such as viral concentration, the size and structure of the encapsulated nucleic acid, container composition, headspace gas, and number of freeze-thaw cycles may all affect the activity of viral compositions.
As a consequence, the utility of many viruses in biopharmaceutical preparations is often limited by the instability of compositions of the viruses, particularly upon storage. For example, even when viral compositions are stored at very low temperature (for example −80° C.) in the frozen state, a significant loss of infectivity may still occur over time. A further loss of infectivity may occur upon thawing of the frozen viral composition.
In addition, as low temperature storage conditions are not always available, it would be advantageous to develop formulations that improve the preservation of frozen viral formulations above −80° C. for extended periods of time, such as extended storage at temperatures just below freezing. Indeed, viral compositions that must be stored at very low temperature and cannot be stored at standard freezer temperatures (for example −10° C. to −20° C.) for substantial periods of time represent a serious impediment to the widespread clinical use of many viruses.
As will be also appreciated, the storage of products at standard freezer temperatures may also be problematic, because often such freezers undergo temperature cycling that may result in the viral composition being subjected to temperatures above freezing, and as such the compositions may undergo repeated cycles of freezing and thawing. Freeze-thawing may also occur during large scale production, handling or distribution.
It would also be advantageous to develop viral compositions that can maintain the desired pH of the composition for extended periods of time despite being exposed to refrigeration temperatures and/or subjected to conditions such as freeze-thawing, especially the slow rate of freeze-thawing that may occur during large scale production, handling or distribution.
Finally, increasingly high concentrations of virus are also being required for therapeutic purposes. However, the concentration of virus in a composition may present additional problems to the ability to preserve a virus. In particular, a high concentration of virus may contribute significantly to viral instability due to aggregation and/or precipitation.
Therefore for many viruses a deficiency has been the inability to formulate compositions that acceptably preserve the virus, particularly in the frozen state. Such deficiencies with the ability to preserve the activity of viral compositions often preclude their use for gene therapy, for vaccination, or for other purposes.
It is therefore an aim of the present invention to provide a composition for the improved preservation of viruses.
Throughout this specification reference may be made to documents for the purpose of describing various aspects of the invention. However, no admission is made that any reference cited in this specification constitutes prior art. In particular, it will be understood that the reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in Australia or in any other country. The discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinency of any of the documents cited herein.